Oligonucleotides with inverted polarity

ABSTRACT

Oligonucleotides having tandem sequences of inverted polarity, i.e., oligonucleotides comprising regions of the formula:   3&#39; - - - - - 5&#39; - - C - - 5&#39; - - - - - 3&#39; (1)  or  5&#39; - - - - - 3&#39; - - C - - 3&#39; - - - - - 5&#39; (2)  wherein -C- symbolizes any method of coupling the nucleotide sequence of opposite polarity, are useful for forming an extended triple helix with a double-helical nucleotide duplex. The inverted polarity also stabilizes the single-strand oligonucleotides to exonuclease degradation.

This is a continuation-in-part of U.S. patent application Ser. No.502,272, filed 29 Mar. 1990, abandoned, which is a continuation-in-partof U.S. patent application Ser. No. 425,803, filed 23 Oct. 1989,abandoned.

TECHNICAL FIELD

The invention is directed to oligonucleotides having tandem sequences ofinverted polarity . These oligonucleotides are useful for forming triplehelices with double-stranded duplex DNA. The oligomers with invertedpolarities allow a first sequence of the oligonucleotide to bind to asegment on one strand of a target duplex, and a second sequence withinverted polarity sequence to bind to a segment on the other strand ofthe duplex. Also, the invention oligonucleotides may be stabilized bythis inversion of the polarity which presents an unnatural terminus orinternal linkage, thereby avoiding potential damage by nucleases.

BACKGROUND ART

Oligonucleotides are known to be able to bind to double-helical DNA inthe major groove of the helix, thereby forming a triple helix structure.The code for binding to form a triple helix (hereinafter referred to asthe triple helix code) does not follow the same code as that for thebinding of two single-stranded polynucleotides to form a double helix.The code for triple helix formation is set forth, for example, in Moser,H. E. and Dervan, P. B., Science (1987) 238: 645-650, and in Coohey, M.,et al., Science (1988) 241:456-459. According to this code, isomorphousbase triplets (T-A-T and C-G-C⁺) can be formed between anyhomopurine-homopyrimidine duplex and a corresponding, third,homopyrimidine strand. Since the rules limit the sequences which canform a triple helix it would be desirable to obtain oligonucleotideswhich can bind to form a triple helix in one portion of a duplex andcross over the groove in the double helix to bind to another portion ofthe duplex, thereby extending the portion of the duplex which can be"read." Such oligonucleotides would allow for identification andlocation of unique portions of double-helical DNA similar to the mannerin which unique portions of single-stranded DNA are identified andlocated by single-stranded oligonucleotides.

The invention provides oligonucleotides which have inverted polaritiesfor at least two regions of the oligonucleotide, thereby allowing therespective inverted polarity segments to read complementary strands of adouble-helical duplex. By "inverted polarity" is meant that theoligonucleotide contains tandem sequences which have opposite polarity,i.e., one having polarity 5'→3' followed by another with polarity 3'→5',or vice versa. This implies that these sequences are joined by linkageswhich can be thought of as effectively a 3'--3' internucleotidejunction, (however the linkage is accomplished), or effectively a 5'--5'internucleotide junction. Such oligomers have been suggested asby-products of reactions to obtain cyclic oligonucleotides byCapobianco, M. L. et al., Nucleic Acids Res (1990) 18: 2661-2669.

DISCLOSURE OF THE INVENTION

The ability of oligonucleotide sequences to hybridize to double-strandedduplex DNA is enhanced by providing oligonucleotides with invertedpolarity so that the binding oligonucleotide can skip from onecomplementary strand in the duplex to the other as its polarity shifts.In its simplest embodiment, there is a single inversion of polarity inthe binding oligonucleotide; of course, inversions can be inserted inany arbitrary number depending upon the number of "switchbacks" desired.

Thus, in one aspect, the invention is directed to "switchback"oligonucleotide sequences containing at least two tandem sequences ofopposite polarities and thus at least one linkage which inverts thepolarity of the oligonucleotide, and to methods of preparing and usingthese oligonucleotides.

The invention also comprises a method for binding a switchbackoligonucleotide to portions of both strands of a double-helicalpolynucleotide duplex comprising the step of hybridizing thedouble-helical polynucleotide duplex with an oligonucleotide to form atriplex wherein the oligonucleotide is characterized by a first sequenceof nucleotides capable to bind a portion of the first strand of theduplex, followed by a second sequence of nucleotides having oppositepolarity capable to bind a portion on the second strand of the duplexwhich is proximal to said target portion on the first strand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a 1,4-dihydroxylmethylbenzene-mediated linkage between twoadjacent ribosyl residues and two adjacent xylosyl residues.

FIG. 2 shows an cutline of the reaction scheme to include a dimersynthon in the oligomer to obtain a switchback.

MODES OF CARRYING OUT THE INVENTION

As used herein "oligonucleotide" is generic to polydeoxyribonucleotides(containing 2'-deoxy-D-ribose or modified forms thereof), i.e., DNA, topolyribonucleotides (containing D-ribose or modified forms thereof),i.e., RNA, and to any other type of polynucleotide which is anN-glycoside or C-glycoside of a purine or pyrimidine base, or modifiedpurine or pyrimidine base. The term "nucleoside" or "nucleotide" willsimilarly be generic to ribonucleosides or ribonucleotides,deoxyribonucleosides or deoxyribonucleotides, or to any other nucleosidewhich is an N-glycoside or C-glycoside of a purine or pyrimidine base,or modified purine or pyrimidine base. Thus, the stereochemistry of thesugar carbons may be other than that of D-ribose in certain limitedresidues, as further described below.

"Nucleoside" and "nucleotide" include those moleties which contain notonly the known purine and pyrimidine bases, but also heterocyclic baseswhich have been modified. Such modifications include alkylated purinesor pyrimidines, acylated purines or pyrimidines, or other heterocycles.Such "analogous purines" and "analogous pyrimidines" are those generallyknown in the art, many of which are used as chemotherapeutic agents. Anexemplary but not exhaustive list includes 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromourcil,5-carboxymethylamino-methyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyl-adenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5'methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, wybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosinne, and2,6-diaminopurine. "Nucleosides" or "nucleotides" also include thosewhich contain modifications in the sugar moiety, for example, whereinone or more of the hydroxyl groups are replaced with halogen, aliphaticgroups, or functionalized as ethers, amines, and the like. Examples ofmodified nucleosides or nucleotides include, but are not limited to:

    ______________________________________                                        2-aminoadenosine 2'-deoxy-2-aminoadenosine                                    5-bromouridine   2'-deoxy-5-bromouridine                                      5-chlorouridine  2'-deoxy-5-chlorouridine                                     5-fluorouridine  2'-deoxy-5-fluorouridine                                     5-iodouridine    2'-deoxy-5-iodouridine                                       5-methyluridine  (2'-deoxy-5-methyluridine                                                     is the same as thymidine)                                    inosine          2'-deoxy-inosine                                             xanthosine       2-deoxy-xanthosine                                           ______________________________________                                    

Furthermore, as the α anomer binds to duplexes in a manner similar tothat for the β anomers, one or more nucleotides may contain this linkageor a domain thereof. (Praseuth, D., et al, Proc Natl Acad Sci (USA)(1988) 85:1349-1353).

The switchback oligonucleotides of the present invention may be of anylength, but lengths of greater than or equal to about 10 nucleotides,and preferably greater than about 15, are preferred. However, the longeroligonucleotides may also be made, particularly those of greater than 50nucleotides or greater than 100 nucleotides. Oligonucleotides maycontain conventional internucleotide phosphodiester linkages or maycontain modified forms such as phosphoramidate linkages. Thesealternative linking groups include, but are not limited to embodimentswherein a moiety of the formula P(O)S, P(O)NR₂, P(O)R, P(O)OR', CO, orCONR₂, wherein R is H (or a salt) or alkyl (1-12C) and R' is alkyl(1-6C) is joined to adjacent nucleotides through --O-- or --S--. Not allsuch linkages in the same oligomer need to be identical.

Inversions of polarity can also occur in "derivatives" ofoligonucleotides. "Derivatives" of the oligomers include thoseconventionally recognized in the art. For instance, the oligonucleotidesmay be covalently linked to various moleties such as intercalators,substances which interact specifically with the minor groove of the DNAdouble helix and other arbitrarily chosen conjugates, such as labels(radioactive, fluorescent, enzyme, etc.). These additional moieties maybe derivatized through any convenient linkage. For example,intercalators, such as acridine can be linked through any available --OHor --SH, e.g., at the terminal 5' position of RNA or DNA, the 2'positions of RNA, or an OH, NH₂, COOH or SH engineered into the 5position of pyrimidines, e.g., instead of the 5 methyl of cytosine, aderivatized form which contains, for example, --CH₂ CH₂ NH₂, --CH₂ CH₂CH₂ OH or --CH₂ CH₂ CH₂ SH in the 5 position. A wide variety ofsubstituents can be attached, including those bound through conventionallinkages. The indicated --OH moleties in the oligomers may be replacedby phosphonate groups, protected by standard protecting groups, oractivated to prepare additional linkages to other nucleotides, or may bebound to the conjugated substituent. The 5' terminal OH may bephosphorylated; the 2'--OH or OH substituents at the 3' terminus mayalso be phosphorylated. The hydroxyls may also be derivatized tostandard protecting groups.

The segments of 5'→3' or 3'→5' polarity are conventionally synthesized.Methods for such synthesis are found, for example, in Froehler, B., etal., Nucleic Acids Research (1986) 14:5399-5467; Nucleic Acids Research(1988) 16:4831-4839; Nucleosides and Nucleotides (1987) 6:287-291.Froehler, B., Tet Lett (1986) 27:5575-5578; and copending Ser. No.248,517, filed Sep. 23, 1988, incorporated herein by reference.

In general, there are two commonly used solid phase-based approaches tothe synthesis of oligonucleotides containing conventional 3'→5' or 5'→3'linkages, one involving intermediate phosphoramidites and the otherinvolving intermediate phosphonate linkages. In the phosphoramiditebased synthesis, a suitably protected nucleotide having acyanoethylphosphoramidite at the position to be coupled is reacted withthe free hydroxyl of a growing nucleotide chain derivatized to a solidsupport. The reaction yields a cyanoethylphosphite, which linkage mustbe oxidized to the cyanoethylphosphate at each intermediate step, sincethe reduced form is unstable to acid. The H-phosphonate-based synthesisis conducted by the reaction of a suitably protected nucleosidecontaining an H-phosphonate moiety at a position to be coupled with asolid phase-derivatized nucleotide chain having a free hydroxyl group,in the presence of a suitable activator to obtain an H-phosphonatediester linkage, which is stable to acid. Thus, the oxidation to thephosphate or thiophosphate can be conducted at any point during thesynthesis of the oligonucleotide or after synthesis of theoligonucleotide is complete. The H-phosphonates can also be converted tophosphoramidate derivatives by reaction with a primary or secondaryamine in the presence of carbon tetrachloride. To indicate the twoapproaches generically, the incoming nucleoside is regarded as having an"activated phosphite/phosphate" group.

Variations in the type of internucleotide linkage are achieved by, forexample, using the methyl phosphonate precursors rather than theH-phosphonates per se, using thiol derivatives of the nucleosidemoleties and generally by methods known in the art. Non-phosphorousbased linkages may also be used, such as the formacetyl type linkagesdescribed and claimed in co-pending applications U.S. Ser. Nos. 426,626and 448,914, filed on 24 October 1989 and 11 December 1989, bothassigned to the same assignee and both incorporated herein by reference.

Thus, to obtain an oligonucleotide segment which has a 3'→5' polarity, anucleotide protected at the 5' position and containing an activatedphosphite/phosphate group at the 3' position is reacted with thehydroxyl at the 5' position of a nucleoside coupled to a solid supportthrough its 3'-hydroxyl. The resulting condensed oligomer is deprotectedand the reaction repeated with an additional 5'-protected,3-'phosphite/phosphate activated nucleotide. Conversely, to obtain anoligomeric segment of 5'→3' polarity a nucleotide protected in the 3'position and containing an activated phosphite/phosphate in the 5'position is reacted with a nucleotide oligomer or nucleoside attached toa solid support through the 5' position, leaving the 3'-hydroxylavailable to react. Similarly, after condensation of the incomingnucleotide, the 3' group is deprotected and reacted with an additional3'-protected, 5'-activated nucleotide. The sequence is continued untilthe desired number of nucleotides have been added.

In addition to employing these very convenient and now most commonlyused, solid phase synthesis techniques, oligonucleotides may also besynthesized using solution phase methods such as triester synthesis.These methods are workable, but in general, less efficient foroligonucleotides of any substantial length.

In their most general form, the inverted polarity oligonucleotides ofthe invention contain at least one segment along their length of theformula: ##STR2## where --C-- symbolizes any method of coupling thenucleotide sequences of opposite polarity.

In these formulas, the symbol 3'- - - 5' indicates a stretch of oligomerin which the linkages are consistently formed between the 5' hydroxyl ofthe ribosyl residue of the nucleotide to the left with the 3' hydroxylof the ribosyl residue of the nucleotide to the right, thus leaving the5' hydroxyl of the rightmost nucleotide ribosyl residue free foradditional conjugation. Analogously, 5'- - - 3' indicates a stretch ofoligomer in the opposite orientation wherein the linkages are formedbetween the 3' hydroxyl of the ribosyl residue of the left nucleotideand the 5' hydroxyl of the ribosyl residue of the nucleotide on theright, thus leaving the 3' hydroxyl of the rightmost nucleotide ribosylresidue free for additional conjugation.

The linkage, symbolized by --C--, may be formed so as to link the 5'hydroxyls of the adjacent ribosyl residues in formula (1) or the 3'hydroxyls of the adjacent ribosyl residues in formula (2), or the"--C--" linkage may conjugate other portions of the adjacent nucleotidesso as to link the inverted polarity strands. "--C--" may represent alinker moiety, or simply a covalent bond.

It should be noted that if the linkage between strands of invertedpolarity involves a sugar residue, either the 3' or 2' position can beinvolved in the linkage, and either of these positions may be in eitherR or S configuration. The choice of configuration will in part determinethe geometry of the oligomer in the vicinity of the linkage. Thus, forexample, if adjacent 3' positions are used to effect a covalent linkage,less severe deformation of the oligonucleotide chain will generallyoccur if both 3' hydroxyls involved in the linkage are in theconventional R configuration. If they are both in the S configuration,this will result in a "kink" in the chain.

In addition to the use of standard oligonucleotide synthesis techniquesor other couplings to effect the 5'--5'or 3'--3' linkage between ribosylmoieties, alternative approaches to joining the two strands of invertedpolarity may be employed. For example, the two appended bases of theopposing termini of the inverted polarity oligonucleotide sequences canbe linked directly or through a linker, or the base of one can be linkedto the sugar moiety of the other. Any suitable method of effecting thelinkage may be employed. The characterizing aspect of the switchbackoligonucleotides of the invention is that they comprise tandem regionsof inverted polarity, so that a region of 3'→5' polarity is followed byone of 5'→3' polarity, or vice versa, or both.

Depending on the manner of coupling the segments with inverted polarity,this coupling may be effected by insertion of a dimeric nucleotidewherein the appropriate 3' positions of each member of the dimer or the5' positions of each member of the dimer are activated for inclusion ofthe dimer in the growing chain, or the conventional synthesis can becontinued but using for the condensing nucleotide a nucleotide which isprotected/activated in the inverse manner to that which would beemployed if the polarity of the chain were to remain the same. Thisadditional nucleotide may also contain a linker moiety which may beincluded before or after condensation to extend the chain.

For example, in one illustrative embodiment of the formulas (1) and (2),these compounds include inversion-conferring linkages of the formulas:##STR3## wherein:

B is any purine or pyrimidine base, modified purine or pyrimidine base,or any desired corresponding moiety of an analogous nucleotide and Prepresents a phosphodiester linkage or a conventional substitutetherefor, such as methyl phosphonate;

Y is H, --OR, --SR, --NR₂, O⁻, or S⁻ ;

X is O, S, or NR;

wherein each R is independently H, alkyl (1-12C), aryl(6-12C),aralkyl(7-20C) or alkaryl(7-20C);

n is 0 or 1; and

A is the residue of a linker group.

Thus, in these representations, the bold line refers to the sugar,whereas B represents the bases. The P within the diagonal line in thediagram denotes a phosphodiester or analogous bond. This diagonal linejoins the end of one bold line and the middle of another. Thesejunctions refer to the 5'--OH and 3'--OH, respectively.

This type of linkage is convenient because --C-- can be incorporatedsequentially using the standard solid phase synthesis techniques.Although shown specifically to effect a 5'--5' or 3'--3' linkage, thelinking portion per se can be used to couple sugar-sugar, sugar-base orbase-base on adjacent switchback nucleotide residues. Also, any linkageform can be included using a prelinked dimer in the solid phasesequence.

When n is 0 in the above embodiment, the 3'--3' or 5'--5' linkage issimply formed using standard oligonucleotide synthesis techniqueswherein the nucleotide to be added to the sequence is protected andactivated in the opposite orientation from that which would be used ifthe original chain polarity were followed. When n=1, a linker isutilized to effect the inverted polarity linkage. There is notheoretical reason that n cannot be >1; however, generally it is moreconvenient to limit the synthesis to the intermediation of one linker.

When a linker moiety is employed, the phosphite/phosphate activatedlinker can be included directly in the continuing oligonucleotidesynthesis, followed by coupling to the first nucleotide of the invertedsequence or the first such nucleotide can be supplied alreadyderivatized to the phosphite/phosphate activated linker. In general, thelinker comprises a diol or diamine, the residue of which appears as "A"in formulas 1' and 2'. Thus, in a typical synthesis protocol, onehydroxyl (or amino) of the diol (or diamine) is protected and the otheris an activated phosphite/phosphate. This protected form can be coupledto the oligonucleotide chain attached to the solid support and thendeprotected and reacted with the subsequent nucleotide residue.

Similar diol or diamine type (or disulfhydryl or hydroxyl/sulfhydryltype) linkers are also convenient when the linkage between invertedpolarity segments is to be effected between adjacent bases or between abase and a sugar moiety, or these can be used to link adjacent sugarsdirectly without the inclusion of the phosphodiester or analog thereof.In these instances, it is generally more convenient to synthesize theswitchback nucleotide dimer independently, and then to insert the dimer,again using standard oligonucleotide synthesis techniques, into theoligonucleotide to be formed. Alternate linker functionalities can beconvenient when adjacent base moleties are to be used, however, ingeneral, convenient forms of linkers are those derived from dihydroxy,diamino (or disulfhydryl or hydroxyl/sulfhydryl) compounds which can besuitably protected and activated so as to integrate them into thestandard oligonucleotide synthesis protocol or otherwise used to obtaininverted dimeric nucleotides.

The significant step in the integration of these linkers, however, isthat the subsequent additions to the oligomer, after the linker isinserted, are activated and deprotected nucleotides having oppositepolarity from that of the preceding portion of the sequence.

Thus, illustrative suitable linkers are or include residues of diols ofthe following formulas or their analogous diamines (or alcohol amines).For ease of representation, the diol structures are used, but it shouldbe kept in mind that either or both hydroxyl functionality may bereplaced by an amino group or a sulfhydryl group.

HO(CH₂)_(n1) OH, wherein n1 is an integer that is usually 1-15, but canalso be in an extended form. One or more of the --CH₂ -- groups may bereplaced by O, S or NH, provided such replacement is not adjacent to aheteroatom. (When integrated into formula 1' or 2', therefore, thislinker will give "A" as a residue of the formula --(CH₂)_(n1) --).

In particular, the diol may represent a polyethylene glycol of theformula HO(CH₂ CH₂ O)_(n2) H, wherein n2 is an integer of 1-5.

The linker may also contain unsaturation, so that it may be of theexemplary formulas:

HOCH₂ (CX₂ CX₂)_(n3) CH₂ OH, wherein n3 is an integer of 1-7 and eachpair of X or adjacent C is independently H or a π bond; or

HOCH₂ (CX₂ CX₂)_(n4) CH₂ (CX₂ CX₂)_(n5) CH₂ OH, wherein n4 and n5 areintegers of 0-7 and wherein the sum of n4 and n5 is not greater than 7and wherein each pair of X or an adjacent C is independently H ortogether are a π bond.

In these embodiments also, one or more methylene groups may be replaced,provided it is not adjacent to an additional heteroatom, by O, S or NH.

The dihydroxy, diamino or equivalent linker compound may also be cyclic,either non aromatic or aromatic. Nonaromatic embodiments include diolssuch as cis- or trans-3-4-dihydroxyfuran, cis- ortrans-2-hydroxymethyl-3-hydroxyfuran, and cis- ortrans-2-hydroxymethyl-4-hydroxyfuran, said furan either furtherunsubstituted, or further substituted with one or two noninterferingalkyl(1-4C) substituents, or may include N-heterocycles such aspiperazine or piperidine.

Linkers containing aromatic rings may include residues of1,2-dihydroxymethylbenzene; 1,4-dihydroxy methylbenzene;1,3-dihydroxymethylbenzene; 2,6-dihydroxy methylnaphthalene;1,5-dihydroxymethylnaphthalene; 1,4-bis(3-hydroxypropenyl)benzene;1,3-bis(3-hydroxy propenyl)benzene; 1,2-bis(3-hydroxypropenyl) benzene;2,6-bis(3- hydroxypropenyl)naphthalene;1,5-bis(3-hydroxypropenyl)naphthalene, 1,4-bis (3-hydroxypropynyl)benzene; 1,3-bis(3-hydroxypropynyl)benzene;1,2-bis(3-hydroxypropynyl) benzene;2,6-bis(3-hydroxypropynyl)naphthalene; and1,5-bis(3-hydroxypropynyl)naphthalene. FIG. 1 shows the coupling using1,4-dihydroxymethylbenzene as it bridges either two ribosyl or twoxylosyl residues.

In addition, the linker may carry additional functional groups, such asanthraquinone and be fairly complex; an example of this type of linkeris: ##STR4##

The length and type of internucleotide linkage at the inverted junctionwill depend in part on the charge concentration (e.g.,polyphosphodiester groups may be too highly concentrated in charge) andon the distance required to span the major groove in the duplex in orderto achieve the required triple helix binding. It is presently consideredthat spanning the two strands of the duplex through a 5'--5' switchbackinvolves no null bases, while a 3'--3' switchback involves 1-4 nullbases in the duplex target. In general, the oligomer spacing accountsfor 0-4 null bases, depending on the embodiment. (A "null" base refersto a base pair in the DNA duplex that does not hydrogen bond to thethird strand moiety.) The length of the linker can be adjustedaccordingly. The proper length and type of linkage may be determined bythose of ordinary skill in the art using routine optimizationprocedures.

Synthesis Methods

For the embodiments of formulas 1' and 2', the synthesis ofoligonucleotides having inverted polarity may be accomplished utilizingstandard solid phase synthesis methods.

This oligonucleotide chain elongation will proceed in conformance with apredetermined sequence in a series of condensations, each one of whichresults in the addition of another nucleotide. Prior to the addition ofa nucleoside having an activated phosphite/phosphate, the protectinggroup on the solid support-bound nucleotide is removed. Typically, forexample, removal of the commonly-employed dimethoxytrityl (DMT) group isdone by treatment with 2.5% v/v dichloroacetic acid/dichloromethane,although 1% w/v trichloroacetic acid/dichloromethane or ZnBr₂ -saturatednitromethane, are also useful. Other deprotection procedures suitablefor other protecting groups will be apparent to those of ordinary skillin the art. The deprotected nucleoside or oligonucleotide bound to solidsupport is then reacted with the suitably protected nucleotidecontaining an activated phosphite/phosphate. After each cycle thecarrier-bound nucleotide is preferably washed with anhydrouspyridine/acetonitrile (1:1, v/v), again deprotected, and thecondensation reaction is completed in as many cycles as are required toform the desired number of congruent polarity internucleotide bondswhich will be converted to phosphoramidates, phosphorodithioates,phosphorothioates or phosphodiesters as desired.

In one embodiment, to provide the switchback, the incoming activated,protected nucleoside is provided in the opposite polarity to thesupport-bound oligomers. Thus, for example, where the support-boundoligomer is 3'→5', the deprotected 5' hydroxyl is reacted with a3'-protected, 5'-activated monomer, and the synthesis continued withmonomers activated at the 5' position and protected at the 3' position.

In another embodiment, to provide a linker in the switchback, a moleculehaving one end which is activated for condensation (such as a hydrogenphosphonate) to the support-bound oligonucleotide and another end whichis a protected hydroxyl group (or protected thio group) is condensedonto the support-bound oligonucleotide. The linker group is condensedand deprotected using the same conditions as those used to condense anddeprotect the protected nucleoside hydrogen phosphonate. Subsequentextension of the oligonucleotide chain then uses oligonucleotideresidues which are activated and protected in the opposite manner fromthose used to synthesize the previous portion of the chain.

If coupling of the inverted portion of the oligonucleotide is through aninternucleotide linkage conjugating the bases of adjacent nucleotides orthe base of one nucleotide to the ribosyl moiety of the other, oradjacent ribosyl residues through linkages which do not involveactivated phosphite/phosphate, it is preferable to form the dimericnucleotide, which is then included in the synthesis in suitablyactivated and protected form. For example, adjacent methyl cytosines orthymidines may be linked through the methyl groups at the 5-positions ofthe pyrimidine rings using a variety of techniques by converting the5-position to, for example, hydroxymethyl, allyl amine, acrylic acid, orpropenyl residues, as is commonly practiced. These reactive groups canthen be further coupled through bifunctional linkers or by suitablealternate condensation to obtain dimeric forms of the methyl cytidine orthymidine, or mixed nucleosides. For inclusion of the dimer in theoligonucleotide of inverted polarity, the dimer is protected, if needed,in, for example, both 5' positions and activated in one 3' position andprotected in the other for continuation of the synthesis. Extension ofthe chain continues from the included dimer using nucleosides ofinverted protection/activation patterns.

One series of reactions, resulting in linkage of the 3' positions inadjacent xylosyl residues through 1,4-dimethoxybenzene is shown inReaction Scheme 1. ##STR5##

In another example, for a dimer wherein adjacent 5-positions of thebases are linked through --(CH═CH--CH₂ NH)₂ CO, the inclusion of thisdimer to obtain a 5'--5' link can be shown diagrammatically in FIG. 2where S= polymeric or other solid phase support, Pr_(B) =a DMTprotecting group; Pr_(C) is a trimethyl acetyl protecting group; P_(A)=activated phosphite/phosphate; and P is as defined above.

Dimers may also be formed between adjacent sugars, and the resultingdimers used as above in standard synthesis. For example, the 3'positions of two ribosyl, xylosyl or ribosyl/xylosyl moieties onadjacent nucleotides may be linked through a p-dihydroxymethyl benzeneor other linker such as 1,3-propylene glycol or ddR of the formula:##STR6## and the 5' positions of the dimer used in subsequent synthesis.(For these latter two linkers, standard incorporation into the oligomersynthesis scheme can also be used.) In this case one 5'-position isprotected with a DMT and the other is activated phosphite/phosphate.Conversely, for adjacent sugars linked through the 5' position, one3'-position is protected and the other activated. As stated above, thegeometry of the oligonucleotide at the linkage site will be affected bythe chirality of the 3' carbons involved in the linkage.

As stated above, all of the internucleotide linkages in the resultingoligomer need not be identical. By use of appropriate synthesistechniques, some can be phosphodiesters, some phosphonates, somephosphoramidates, etc.

As set forth above, the inverted polarity oligonucleotides of thisinvention may be derivatized. One convenient method to form suchderivatization is through the phosphoramidate linkage. The amine whichis utilized to form the phosphoramidate may employ substituents that canconfer useful properties to the oligonucleotide. For example, if anamine linked to a polyethylene glycol, a polypeptide or a lipophilicgroup is utilized, such a group may facilitate transport of theoligonucleotide through the cell membranes, thus increasing the cellularuptake of the oligonucleotide. A substituent on the amine may alsoinclude a group which affects the target DNA to which theoligonucleotide will bind such as providing covalent linkages to thetarget strand to facilitate cleavage or intercalation of the switchbackoligonucleotide to the target strand. The substituents on the amine maycontain chromophoric groups such as fluorescein or other labels,including radioactive labels, chelating agents and metal chelated ions,to label the oligonucleotide for identification. The substituents maythus also serve a cutting function (i.e., a site for cutting the duplex)or a receptor function such as a receptor ligand. The substituents onthe amine which form the phosphoramidate linkage may thus be virtuallyany moiety which does not prevent the oligonucleotide from binding tothe target duplex.

More than one derivatizing moiety may also be used as two or morephosphoramidate linkages need not contain the same substituents. Thismay be accomplished by generating a first nucleotide hydrogenphosphonate linkage and then oxidizing it with a first amine, generatinga second hydrogen phosphonate linkage and oxidizing it with a second(different) amine.

While the formation of the phosphoramidate linkage provides a convenientmethod for attaching the groups which derivatize the oligonucleotide toconfer useful properties, other methods may also be used. The usefulsubstituents may be attached to the sugar moieties or to the bases, orby any other method generally known in the art.

After completion of the synthesis, the oligonucleotide is separated fromthe carrier using conventional methods, such as, by incubation withconcentrated ammonium hydroxide. Any protecting groups (for example, onthe purine or pyrimidine bases) may be removed using, for example, theconventional concentrated ammonia reagent. The oligonucleotide is thenpurified by conventional means such as HPLC, PAGE (polyacrylamide gelelectrophoresis) or any other conventional technique.

It will be understood that while the above method has been described inconnection with use of a solid state carrier, it is also possible toconduct the synthesis without the use of a solid state support. In suchan event, in place of the support a 3'-hydroxy protecting group which isdifferent from the 5' protecting group used in the course of thecondensation, may be utilized so that the 5' protecting group may beselectively removed while the 3' protecting group remains intact.

Binding Properties

The oligonucleotides with inverted polarity according to the presentinvention are useful to bind to a double-helical nucleotide duplex toform a triplex. The conditions for hybridization to form a triplex areknown, as shown for example by Moser and Dervan, supra. Hybridizationnormally takes place at a pH in the range of about 6-7. The nucleotidesaccording to the present invention will comprise, on one side of the3'--3' (or 5'--5') inversion, bases which bind to one strand of theduplex according to the triple helix code, with the bases on the otherside of the 3'--3' (or 5'--5') junction selected to be bases which willbind according to this code to the subsequent bases on the oppositestrand of the duplex.

In this manner triple helix recognition may be extended by switchingrecognition from one strand of the duplex to the other and then backagain, if desired. Also, certain nucleases may be blocked, since theoligonucleotides according to the present invention can present ends notrecognizable by exonucleases. Thus, oligonucleotides having two 5'-ends,will be resistant to 3'-exonucleases.

Since the switchback oligonucleotides of the invention are intended toexpand the strength of binding to duplex DNA, the sequence ofnucleotides in each portion of the oligonucleotide is determined by thesequence of bases in the target duplex. Target duplex sequences whichcontain multiple adenyl residues in a homopurine region of one chain,followed by a region of homopurines comprising guanines in the oppositestrand will mandate a switchback oligonucleotide which is polyT in thepolarity opposite to the polyA tract followed by polyC in the polarityopposite to that of the polyG tract, assuming antiparallel binding.Alternating A/G sequences in the first strand of the target duplex willmandate alternating T/C sequences in a first region of congruentpolarity (same sense) in the switchback oligonucleotide followed by asequence of inverted polarity which matches the second strand sequentialsequence in the duplex, again assuming antiparallel binding. As theswitchback oligonucleotide is intended for complexing to form a triplehelix, it is generally comprised mainly of pyrimidine-based nucleotides.However, it is also known that the geometry of the double helix resultsin a spacing requirement so that at a 3'--3' linkage there will beapproximately 0-4 essentially null bases in the oligonucleotide; thereappear to be no null bases required in the 5'--5' switchback. The nullbase spacing can be provided by arbitrary nucleotide insertions or,alternatively, the length of a linker moiety may be adjusted tocompensate for this.

In addition to enhanced capability to bind to the duplex by formation ofa triplex through the switchback in the oligonucleotide of theinvention, these oligonucleotides may also form D-loops with the duplex.In this situation, the region of a first polarity may, for example, forma triplex, while the inverted portion displaces a section of one strandof the duplex to result in a substitute duplex in the relevant region.The design of the sequence of bases in the oligonucleotide takes accountof this by utilization of a sequence which is designed to base pairhybridize to the target strand.

In summary, the switchbacks may bind the duplexes in a variety ofconformations including both antiparallel and parallel binding modes,direct crossovers of the form ##STR7## switchback modes of the form##STR8## or D-loops.

Utility and Administration

As the switchback oligonucleotides of the invention are capable ofsignificant duplex binding activity, these oligonucleotides are usefulin oligonucleotide therapy. Oligonucleotide therapy is a generic termwhich includes the use of specific binding oligonucleotides toinactivate undesirable DNA or RNA sequences in vitro or in vivo. Becauseof their superior binding ability to duplex DNA, the switchbackoligonucleotides are particularly helpful in this regard.

Most diseases and other conditions are characterized by the presence ofundesired DNA or RNA, some of which may be in duplex form. Thesediseases and conditions can be treated using the principles ofoligonucleotide therapy as is generally understood in the art.Oligonucleotide therapy includes targeting a specific DNA or RNAsequence through complementarity or through any other specific bindingmeans, in the case of the present invention by sequence-specificorientation in the major groove of the DNA double helix.

In addition to oligonucleotide therapy applications, wherein specificsequence recognition is significant, alternate therapeutic mechanismsfor the switchback oligomers of the invention can be advantageouslyemployed. Such oligomers are generally useful as inhibitors ofpolymerases such as vital polymerases, to interfere with binding factorsto nucleic acids such as transcription initiating or inhibiting factors,to induce the production of interferon endogenously, and so forth. Theoligomers of the invention may be administered singly, or combinationsof oligomers may be administered for adjacent or distant targets or forcombined effects of antisense mechanisms with the foregoing generalmechanisms.

In therapeutic applications, the switchback oligomers are utilized in amanner appropriate for antisense therapy in general or to take advantageof the alternate therapeutic mechanisms set forth above. For suchtherapy, the oligomers can be formulated for a variety of modes ofadministration, including systemic, topical or localized administration.Techniques and formulations generally may be found in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Penna., latestedition. The oligomer active ingredient is generally combined with acarrier such as a diluent or excipient which may include fillers,extenders, binders, wetting agents, disintegrants, surface-activeagents, or lubricants, depending on the nature of the mode ofadministration and dosage forms. Typical dosage forms include tablets,powders, liquid preparations including suspensions, emulsions andsolutions, granules, capsules and suppositories, as well as liquidpreparations for injections, including liposome preparations.

For systemic administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the oligomers of the invention are formulated in liquidsolutions, preferably in physiologically compatible buffers such asHank's solution or Ringer's solution. In addition, the oligomers may beformulated in solid form and redissolved or suspended immediately priorto use. Lyophilized forms are also included.

Systemic administration can also be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, bile sales and fusidic acidderivatives for transmucosal administration. In addition, detergents maybe used to facilitate permeation. Transmucosal administration may bethrough use of nasal sprays, for example, or suppositories. For oraladministration, the oligomers are formulated into conventional oraladministration forms such as capsules, tablets, and tonics.

For topical administration, the oligomers of the invention areformulated into ointments, salves, gels, or creams, as is generallyknown in the art.

In addition to use in therapy, the oligomers of the invention may beused as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to which they specifically bind. Suchdiagnostic tests are conducted by hybridization through triple helixformation which is then detected by conventional means. For example, theoligomers may be labeled using radioactive, fluorescent, or chromogeniclabels and the presence of label bound to solid support detected.Alternatively, the presence of a triple helix may be detected byantibodies which specifically recognize these forms. Means forconducting assays using such oligomers as probes are generally known.

In addition to the foregoing uses, the ability of the oligomers toinhibit gene expression can be verified in in vitro systems by measuringthe levels of expression in recombinant systems.

The following examples are provided to illustrate but not to limit theinvention.

EXAMPLE 1 Preparation of 3'-DMT-N⁴ -benzoyl-dC-5'-H-phosphonate

6.4 g (1C mmole) of 5'-DMT N⁴ -benzoyl deoxy-C is dried from 100 ml ofpyridine, dissolved into 100 ml of pyridine and to this is added 4 g(11.8 mmole) of DMT-Cl and the reaction mixture stirred at roomtemperature for three days.. The reaction mixture is evaporated toapproximately half the volume and diluted with 100 ml of CH₂ Cl₂, washwith 5% sodium bicarbonate (2×100 ml), dry over sodium sulfate andevaporate to dryness. The crude mixture is dissolved into 100 ml oftoluene and evaporated to a foam, and this is repeated one more time.The solid is taken up in 50 ml of diethyl ether/50 ml of CH₂ Cl₂ andprecipitated into 900 ml of hexane at room temperature. The solid isisolated and dissolved into 15 ml of CH₂ Cl₂, cool to 0° C. and add 100ml of saturated ZnBr₂ in isopropanol/CH₂ Cl₂ (15/85) and stirred for 15minutes. Reaction mixture is quenched into 400 ml of 1M NH₄ OAc, theorganic layer separated and wash with NaHCO₃ (1×200 ml), dry over Na₂SO₄ and evaporate. Purify by silica gel chromatography (CH₂ Cl₂ /5%MeOH) to yield 50% of the 5'--OH product. The 5'--OH nucleoside is driedfrom 50 ml of pyridine then taken up in 10 ml pyridine and 10 ml ofmethylene chloride, to which is added 2 eq. of 1M PA/CH₂ Cl₂ in 5 ml ofpyridine. This mixture is stirred at r.t. for 15 minutes and quenchedinto 1M TEAB, the layers are separated, and the organic layer is washedwith TEAB (1 time), dried over sodium sulfate and evaporated to dryness.The reagent PA is yon Boom's Reagent,2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one. The nucleosideH-phosphonate is purified by silica gel chromatography (1%triethylamine/CH₂ Cl₂) with a MeOH gradient.

EXAMPLE 2 Preparation of 3'-DMT-thymidine-5'-H-phosphonate

1.2 g (5 mmole) of thymidine is dried from 50 ml of pyridine, taken upin 20 ml of pyridine and under Ar is added 820 mg (5.5 mmole) oft-butyldimethyl silyl chloride in 5 ml of pyridine. The mixture isstirred at room temperature for one day, concentrated to approximately10 ml, diluted with 75 ml of CH₂ Cl₂, washed with 5% NaHCO₃, backextracted the aqueous with CH₂ Cl₂, dried over Na₂ SO₄ and evaporated.The crude nucleoside is dried from 20 ml pyridine, taken up in 30 mlpyridine and to it is added 1.7 g (5 mmole) of DMT-Cl and 0.4 ml oftriethylamine, after which the mixture is stirred for three days. Afterevaporation to approximately 10 ml, the mixture is diluted with 75 ml ofCH₂ Cl₂ and washed with NaHCO₃ (2×100 ml), dried over Na₂ SO₄ andevaporated. The 5'--OH nucleoside is taken up into 60 ml THF and 20 mlof 1M TBAF/THF is added. After stirring for one hour, the mixture isevaporated to an oil, taken up in CH₂ Cl₂ and applied to silica gelcolumn. Yield 50%. The 5'--OH nucleoside is then converted toH-phosphonate as in Example 1.

EXAMPLE 3 Synthesis of Oligomer-Containing Switchbacks

Polynucleotide H-phosphonates condensed at the 3'-end to a solid polymersupport are prepared as described by Froehler, et al., Nuc Acids Res16:4831-4839 (1988); Nuc Acids Res 14:5399-5467 (1986); and Nucleosidesand Nucleotides 6:287-291 (1987); using the DBU salt of 5'-protectednucleoside H-phosphonates. After four couplings, one coupling cycle isperformed using the ethylene glycol derivative: ##STR9## Thepolynucleotide H-phosphonate is then oxidized with aqueous I₂ (0.1M inN-methyl morpholine/water/THF, 5/5/90) to form internucleotide diesterlinkages. Then five coupling cycles are performed using 3'-protectednucleoside 5'-H-phosphonates, prepared as in Examples 1 and 2. Afterthese couplings the remaining H-phosphonate linkages on thepolynucleoside are oxidized with 2-methoxyethylamine in Pyr/CCl₄(1/5/5), to generate a 10-mer with five diester linkages (one of whichis with the ethylene glycol linker) and five phosphoramidate linkages(one of which is with the ethylene glycol linker). The oligomer isremoved from the solid support, deprotected with concentrated NH₄ OH,purified by HPLC (PRP) using an acetonitrile gradient in 50 mM aqueousTEAP. DMT is removed using 80% HOAc (R.T.) and the solvent isevaporated. The product is desalted, and isolated by evaporation.

Thus, in this manner the following are prepared, wherein P₁ represents##STR10## (having an ionization state determined by pH): ##STR11##

By utilizing nucleosides derivatized to solid support through the 5'portion and extending the chain with the 3'-protected, 5'-activatednucleosides of Examples 1 and 2, followed by coupling to DMT--O--CH₂ CH₂--P₁ as above, followed by chain extension, with conventional5'-protected, 3'-activated nucleosides, the following are prepared.##STR12##

By insertion of an additional linker, the following are prepared:##STR13##

EXAMPLE 4 Synthesis and Assay of a Recognition Switchback

Using the methods of Example 3, a switchback 24-mer linked withpropylene glycol was prepared of the formula: ##STR14##

The duplex DNA to which this oligomer binds has a 26-bp target regionhaving 2 null bases to accommodate the switchback. This target region isof the formula: ##STR15##

To assess the capacity of the switchback oligomer to bind the targetregion, an assay was conducted as follows. A 144-bp fragment containingfour 36-bp cassettes, one of which corresponds to the desired targetregion, was cloned into pTZ18U, a commercially available cloning vector.The plasmid was cleaved and labeled at both ends with 32 PdCTP, and thendigested with a second restriction enzyme to eliminate the label at oneend and reduce the size of target DNA. The resulting 372 bp fragment waspurified using 5% acrylamide gel.

The switchback oligomer (1 μmol) was incubated with 5×10⁴ cpm labeledtarget in 100 mM NaCl, 10 mM MgCl₂, 50 mM MES, pH 6, at room temperaturefor one hour.

The reaction mixtures were then treated with 0.5 μg/ml DNAse-I for 1minute at room temperature, and extracted to terminate the digestion andremove protein. The DNA was recovered by ethanol precipitation, and thesamples were loaded onto 8% polyacrylamide gel in 80% formamide and rununder denaturing conditions for 2 hours at 60 watts. The gel was driedand placed on film overnight. The results showed that the oligomerprovided protection against DNAse-I activity indicating formation oftriple stranded structures in the target region and not in relatedtarget sequences. Protection is extended over ˜26 bp region, asdetermined with a 14-mer control.

I claim:
 1. An oligonucleotide comprisinga first nucleotide sequencecontaining at least three nucleotide residues, said sequence havingeither 3'→5' or 5'→3' polarity, and, coupled thereto, a secondnucleotide sequence containing at least three nucleotide residues, saidsecond sequence having polarity inverted from that of the firstsequence.
 2. The oligonucleotide of claim 1 whereinthe 5' position of anucleotide at the 5' end of the first sequence is covalently bonded tothe 5' position of the nucleotide at the 5' end of the second sequence,or wherein the 3' position of a nucleotide at the 3' end of the firstsequence is covalently bonded to the 3' position of the nucleotide atthe 3' end of the second sequence.
 3. The oligonucleotide of claim 1whereinthe base of the nucleotide at the 5' end of the first sequence iscovalently bonded to the base of the nucleotide at the 5' end of thesecond sequence, or wherein the base of the nucleotide at the 3' end ofthe first sequence is covalently bonded to the base of the nucleotide atthe 3' end of the second sequence.
 4. The oligonucleotide of claim 1whereinthe base of the nucleotide at the 5' end of the first sequence iscovalently bonded to the 5' position of the nucleotide at the 5' end ofthe second sequence, or wherein the 5' position of the nucleotide at the5' end of the first sequence is covalently bonded to the base of thenucleotide at the 5' end of the second sequence, or wherein the base ofthe nucleotide at the 3' end of the first sequence is covalently bondedto the 3' position of the nucleotide at the 3' end of the secondsequence, or wherein the 3' position of the nucleotide at the 3' end ofthe first sequence is covalently bonded to the base of the nucleotide atthe 3' end of the second sequence.
 5. The oligonucleotide of claim 1wherein said first and second sequences are covalently bonded through alinkage comprising a linker residue wherein said linker residue is of amolecule comprising two functional groups involved in the linkage, eachsaid functional group selected from the group consisting of an amine, asulfhydryl, and a hydroxyl.
 6. The oligonucleotide of claim 5 whereinsaid linker residue is a residue of a diol or diamine.
 7. Theoligonucleotide of claim 6 wherein said diol or diamine is of theformula:HZ(CH₂)_(n1) ZH, wherein each Z is independently O or NH, andwherein n1 is an integer of 1-15 and one or more of said CH₂ groups maybe replaced by O, S, or NH, provided said replacement is not adjacent toa heteroatom.
 8. The oligonucleotide of claim 7 wherein the diol ordiamine is of the formulaHZ(CH₂ CH₂ O)_(n2) H, wherein Z is O or NH andwherein n2 is an integer of 1-5.
 9. The oligonucleotide of claim 6wherein said diol or diamine is of the formulaHZCH₂ (CX₂ CX₂)_(n3) CH₂ZH, wherein each Z is independently O or NH, and wherein n3 is aninteger of 1-7 and each pair of X or adjacent C independently are H ortogether are a π bond.
 10. The diol or diamine of claim 6 wherein saiddiol or diamine is of the formulaHZCH₂ (CX₂ CX₂)_(n4) CH₂ (CX₂ CX₂)_(n5)CH₂ ZH, wherein each Z is independently O or NH, and wherein n4 and n5are integers of 0-7 and wherein the sum of n4 and n5 is not greater than7 and wherein each pair of X or adjacent C independently are H ortogether are a π bond.
 11. The oligonucleotide of claim 6 wherein saiddiol or diamine contains a ring system.
 12. The oligonucleotide of claim11 wherein said ring system is nonaromatic.
 13. The oligonucleotide ofclaim 12 wherein said nonaromatic ring is piperidine, piperazine, furan,tetrahydrofuran, cyclohexene, cyclopentene, cyclopentane or cyclohexane.14. The oligonucleotide of claim 13 wherein the diol is selected fromthe group consisting of cis- or trans-3-4-dihydroxyfuran, cis- ortrans-2-hydroxymethyl-3-hydroxyfuran, and cis- ortrans-2-hydroxymethyl-4-hydroxyfuran, said furan either furtherunsubstituted, or further substituted with one or two noninterferingalkyl(1-4C) substituents.
 15. The oligonucleotide of claim 11 whereinsaid ring system is aromatic.
 16. The oligonucleotide of claim 15wherein said aromatic ring is benzene or naphthalene.
 17. Theoligonucleotide of claim 16 wherein said diol is selected from the groupconsisting of 1,2-dihydroxymethylbenzene; 1,4-dihydroxymethylbenzene;1,3-dihydroxymethylbenzene; 2,6-dihydroxymethylnaphthalene;1,5-dihydroxymethylnaphthalene; 1,4-bis(3-hydroxy propenyl)benzene;1,3-bis(3-hydroxypropenyl)benzene; 1,2-bis (3-hydroxypropenyl)benzene;2,6-bis(3-hydroxypropenyl)naphthalene; 1,5-bis(3-hydroxypropynyl)naphthalene, 1,4-bis(3-hydroxypropynyl)benzene;1,3-bis(3-hydroxypropynyl)benzene; 1,2-bis(3-hydroxypropynyl)benzene;2,6-bis(3-hydroxypropynyl) naphthalene; and1,5-bis(3-hydroxypropynyl)naphthalene.
 18. The oligonucleotide of claim1 wherein said first and second sequence are covalently bonded through alinkage of the formula: ##STR16## wherein: Y is H, --OR, --SR, --NR₂,O³¹ , or S⁻ ;X is O, S, or NR; wherein each R is independently H, alkyl(1-12C), aryl (6-12C), aralkyl (7-20C) or alkaryl (7-20C); n is 0 or 1;and A is the residue of a linker group wherein said residue of a linkergroup is of a molecule comprising two functional groups involved in thelinkage, each said functional group selected from the group consistingof an amine, a sulfhydryl, and a hydroxyl.
 19. The oligonucleotide ofclaim 18 wherein all X are O.
 20. The oligonucleotide of claim 19wherein all Y are O.
 21. The oligonucleotide of claim 18 wherein saidlinker residue, A, is a residue of a diol or diamine.
 22. Theoligonucleotide of claim 21 wherein said linker residue comprises a ringsystem.
 23. The oligonucleotide of claim 22 wherein said ring isaromatic.
 24. The oligonucleotide of claim 23 wherein said aromatic ringis benzene or naphthalene.
 25. The oligonucleotide of claim 24 whereinsaid diol is selected from the group consisting of1,2-dihydroxymethylbenzene; 1,4-dihydroxymethylbenzene;1,3-dihydroxymethylbenzene; 2,6-dihydroxymethylnaphthalene;1,5-dihydroxymethylnaphthalene; 1,4-bis(3-hydroxy propenyl)benzene;1,3-bis(3-hydroxypropenyl)benzene; 1,2-bis(3-hydroxypropenyl)benzene;2,6-bis(3-hydroxypropenyl)naphthalene; 1,5-bis(3-hydroxypropenyl)naphthalene; 1,4-bis(3-hydroxypropynyl)benzene;1,3-bis(3-hydroxypropynyl)benzene; 1,2-bis(3-hydroxypropynyl)benzene;2,6-bis(3-hydroxypropynyl) naphthalene; and1,5-bis(3-hydroxypropynyl)naphthalene.
 26. The oligonucleotide of claim22 wherein said ring is nonaromatic.
 27. The oligonucleotide of claim 26wherein said nonaromatic ring is piperidine, piperazine, furan,tetrahydrofuran, cyclohexene, cyclopentene, cyclopentane or cyclohexane.28. The oligonucleotide of claim 27 wherein the diol is selected fromthe group consisting of cis- or trans-3-4-dihydroxyfuran, cis- ortrans-2-hydroxymethyl-3-hydroxyfuran and cis- ortrans-2-hydroxymethyl-4-hydroxyfuran, said furan either furtherunsubstituted, or further substituted with one or two noninterferingalkyl (1-4C) substituents.
 29. The oligonucleotide of claim 21 whereinsaid diol or diamine is of the formula:HZ(CH₂)_(n1) ZH, wherein each Zis independently O or NH, and wherein n1 is an integer of 1-10 and oneor more of said CH₂ groups may be replaced by O, S, or NH, provided saidreplacement is not adjacent to a heteroatom.
 30. The oligonucleotide ofclaim 29 wherein the diol or diamine is of the formulaHZ(CH₂ CH₂ O)_(n2)H, wherein Z is O or NH and wherein n2 is an integer of 1-5.
 31. Theoligonucleotide of claim 22 wherein said diol or diamine is of theformulaHZCH₂ (CX₂ CX₂)_(n3) CH₂ ZH, wherein each Z is independently O orNH, and wherein n3 is an integer of 1-7 and each pair of X is anadjacent C independently H or a π bond.
 32. The oligonucleotide of claim21 wherein said diol or diamine is of the formulaHZCH₂ (CX₂ CX₂)_(n4)CH₂ (CX₂ CX₂)_(n5) CH₂ ZH, wherein each Z is independently O or NH, andwherein n4 and n5 are integers of 0-7 and wherein the sum of n4 and n5is not greater than 7 and wherein each pair of X is an adjacent Cindependently H or a participant in a π bond.